Amino acid and purine release in rat brain following temporary middle cerebral artery occlusion

Excitatory amino acid release and neurotoxicity in the ischemic brain may be reduced by endogenously released adenosine which can modulate both glutamate or aspartate release and depress neuronal excitability. The present study reports on the patterns of release of glutamate and aspartate; the inhibitory amino acids GABA and glycine; and of the purine catabolites adenosine and inosine from the rat parietal cerebral cortex during 20 and 60 min periods of middle cerebral artery (MCA) occlusion followed by reperfusion. Aspartate and glutamate efflux into cortical superfusates rose steadily during the period of ischemia and tended to increase even further during the subsequent 40 min of reperfusion. GABA release rose during ischemia and declined during reperfusion, whereas glycine efflux was relatively unchanged during both ischemia and reperfusion. Adenosine levels in cortical superfusates rose rapidly at the onset of ischemia and then declined even though MCA occlusion was continued. Recovery to pre-occulusion levels was rapid following reperfusion. Inosine efflux also increased rapidly, but its decline during reperfusion was slower than that of adenosine.     

头孢曲松神经保护作用的研究

目的：探讨头孢曲松通过抑炎通路发挥神经保护作用的机制。方法：45只雄性sD大鼠,随机分为3组（n=15）,预处理组在大鼠大脑中动脉阻断（MCAo）前5d每日给予腹腔注射头孢曲松缓冲液（200mg／kg）,观察局灶性脑缺血2h再灌注24h后,各组间大鼠神经行为学评分、脑梗死容积变化、小胶质细胞和IL-1β的数量变化。结果：再灌注24h后,头孢曲松预处理组可改善大鼠神经行为学评分和脑梗死面积（P〈0．05）,小胶质细胞活化数量减少（P〈0．05）,ELISA检测IL-1β分泌数量有显著下降趋势,但无统计学差异（P=0．18）。结论：头孢曲松可以部分抑制小胶质细胞活化,减少IL-1β释放发挥神经保护作用。     

Anti-ischemic Effect of Curcumin in Rat Brain

Turmeric has been in use since ancient times as a condiment and due to its medicinal properties. Curcumin, the yellow colouring principle in turmeric, is polyphenolic and major active constituent. Besides anti-inflammatory, thrombolytic and anticarcinogenic activities, curcumin also possesses strong antioxidant property. In view of the novel combination of properties, neuroprotective efficacy of curcumin was studied in rat middle cerebral artery occlusion (MCAO) model. Rats were subjected to 2 h of focal ischemia followed by 72 h of reperfusion. They were pre-treated with curcumin (100 mg/kg, po) for 5 days prior to MCAO and for another 3 days after MCAO. The parameters studied were behavioural, biochemical and histological. Treatment with curcumin could significantly improve neurobehavioral performance compared to untreated ischemic rats as judged by its effect on rota-rod performance and grid walking. A significant inhibition in lipid peroxidation and an increase in superoxide dismutase (SOD) activity in corpus striatum and cerebral cortex was observed following treatment with curcumin in MCAO rats as compared to MCAO group. Intracellular calcium levels were decreased following treatment with curcumin in MCAO rats. Histologically, a reduction in the infarct area from 33% to 24% was observed in MCAO rats treated with curcumin. The study demonstrates the protective efficacy of curcumin in rat MCAO model.     

头孢曲松神经保护作用的研究

目的:探讨头孢曲松通过抑炎通路发挥神经保护作用的机制。方法:45只雄性SD大鼠,随机分为3组(n=15),预处理组在大鼠大脑中动脉阻断(MCAO)前5 d每日给予腹腔注射头孢曲松缓冲液(200 mg/kg),观察局灶性脑缺血2 h再灌注24 h后,各组间大鼠神经行为学评分、脑梗死容积变化、小胶质细胞和IL-1β的数量变化。结果:再灌注24 h后,头孢曲松预处理组可改善大鼠神经行为学评分和脑梗死面积(P<0.05),小胶质细胞活化数量减少(P<0.05),ELISA检测IL-1β分泌数量有显著下降趋势,但无统计学差异(P=0.18)。结论:头孢曲松可以部分抑制小胶质细胞活化,减少IL-1β释放发挥神经保护作用。     

Long non-coding RNA RMST silencing protects against middle cerebral artery occlusion (MCAO)-induced ischemic stroke

Long non-coding RNAs (lncRNAs) have emerged as major regulators in neurological diseases, and clarifying their roles in cerebral ischemic injury may provide novel targets for treating ischemic stroke. In this study, we mainly studied the role of lncRNA-RMST in middle cerebral artery occlusion (MCAO)-induced mouse brain injury. We showed that RMST expression level was significantly up-regulated in oxygen-glucose deprivation (OGD)-treated primary hippocampal neuron, MCAO-induced injured brain, and the plasma of patients with ischemic stroke. RMST silencing protected against MCAO-induced ischemic brain injury in vivo and OGD-induced primary hippocampal neuron injury in vitro. Intracerebroventricular injection of RMST shRNA significantly decreased brain RMST expression, reduced brain infarct size, and improved neurological function. Collectively, this study provides evidence that lncRNA is involved in the pathogenesis of ischemic brain injury, and suggests a promising approach of RMST inhibition in treating ischemic stroke.     

改良线栓法大鼠大脑中动脉阻塞模型的建立

目的对大鼠大脑中动脉阻塞（MCAO）再灌注模型进行改良,通过比较再灌注24h时大鼠神经功能评分、梗死率、模型制作时间、成功率和死亡率等指标评价改良线栓法大鼠MCAO再灌注模型的有效性。方法12只SD大鼠随机分为对照和模型两组,对照组采用分离结扎翼腭动脉,从颈外动脉插入线栓至大脑中动脉。模型组采用不分离结扎翼腭动脉,从颈总动脉分叉处插入线栓至大脑中动脉。阻断大脑中动脉血供2h后将线栓拔出实现再灌注。于再灌注24h时观察脑组织组织病理学改变,计算比较两组大鼠神经功能评分、模型制作时间、模型成功率和死亡率以及鼠脑切片TTC染色测量脑梗死率。结果两组MCAO模型在再灌注24h后大鼠神经功能评分、梗死率、模型成功率和死亡率等方面没有显著差异;模型组的模型制作时间显著少于对照组（P〈0．05）。结论采用不分离结扎翼腭动脉,由颈总动脉插入线栓的改良线栓法是稳定和可靠的MCAO造模方法。     

Reduction of Brain Infarction Induced by a Transient Brain Ischemia in mdr1a Knockout Mice

In order to evaluate the functional role of P-glycoprotein (P-gp) in cerebral ischemia, both multidrug resistance 1a knockout (KO) mice and wild-type mice were subjected to transient focal ischemia under a constant body and brain temperature about 37°C. The results showed that the volume of brain infarction induced by middle cerebral artery occlusion in KO mice was significantly smaller than that seen in wild-type mice, although there were no significant differences in cerebral blood flow, physiological data and on anatomical analysis of cerebrovasculature between both groups. We suggest that multidrug resistance 1a P-gp plays a role for adjusting the expressions of endogenous neuronal cell modulating substances, such as cytokines, neuronal peptides, and others, in the brain, which is consistent with a previous paper (Bobrov et al. Neurosci Lett 24: 6–11, 2008).     

Neuronal dependence of extracellular dopamine,acetylcholine, glutamate,aspartate and gamma-aminobutyric acid (GABA) measured simultaneously from rat neostriatum using in vivo microdialysis: reciprocal interactions

Summary The neuronal origin of extracellular levels of dopamine (DA), acetylcholine (ACh), glutamate (Glu), aspartate (Asp) and gamma-aminobutyric acid (GABA) simultaneously collected from the neostriatum of halothane anaesthetized rats with in vivo microdialysis was studied. The following criteria were applied (1) sensitivity to K⁺-depolarization; (2) sensitivity to inhibition of synaptic inactivation mechanisms; (3) sensitivity to extracellular Ca²⁺; (4) neuroanatomical regionality; sensitivity to selective lesions and (5) sensitivity to chemical stimulation of the characterized pathways.It was found that: (1) Extracellular DA levels found in perfusates collected from the neostriatum fulfills all the above criteria and therefore the changes in extracellular DA levels measured with microdialysis reflect actual release from functionally active nerve terminals, and so reflect ongoing synaptic transmission. (2) Changes in neostriatal ACh levels reflect neuronal activity, provided that a ACh-esterase inhibitor is present in the perfusion medium. (3) Extracellular Glu, Asp and GABA could be measured in different perfusion media in the rat neostriatum and probably reflect metabolic as well as synaptic release. However, (4) the majority of the extracellular GABA levels found in perfusates collected from the neostriatum may reflect neuronal release, since GABA levels were increased, in a Ca²⁺-dependent manner, by K⁺-depolarization, and could be selectively decreased by an intrinsic neostriatal lesion. (5) It was not possible to clearly distinguish between the neuronal and the metabolic pools of Glu and Asp, since neostriatal Glu and Asp levels were only slightly increased by K⁺-depolarization, and no changes were seen after decortication. A blocker of Glu re-uptake, DHKA, had to be included in the perfusion medium in order to monitor the effect of K⁺-depolarization on Glu and Asp levels. Under this condition, it was found (6) that neostriatal Glu and Asp levels were significantly increased by K⁺-depolarization, although only increases in the Glu levels were sensitive to Ca²⁺ in the perfusion medium, suggesting that Glu but not Asp is released from vesicular pools. (7) Evidence is provided that selective stimulations of nigral DA cell bodies may lead to changes in release patterns from DA terminals in the ipsilateral neostriatum, which are in turn followed by discrete changes in extracellular levels of GABA and Glu in the same region. Finally, some methodological considerations are presented to clarify the contribution of neuronal release to extracellular levels of amino acid neurotransmitters in the rat neostriatum.     

Neuroprotective effect of curcumin in middle cerebral artery occlusion induced focal cerebral ischemia in rats

Free radical induced neuronal damage is implicated in cerebral ischemia reperfusion (IR) injury and antioxidants are reported to have neuroprotective activity. Several in vitro and in vivo studies have proved the antioxidant potential of curcumin and its metabolites. Hence, in the present study the neuroprotective potential of curcumin was investigated in middle cerebral artery occlusion (MCAO) induced focal cerebral IR injury. 2 h of MCAO and 22 h of reperfusion resulted in the infarct volume of 210.39 +/- 31.25 mm3. Administration of curcumin 100 and 300 mg/kg, i.p. 30 min. after MCAO produced 37.23 +/- 5.10% and 46.39 +/- 10.23% (p < 0.05) reduction in infarct volume, respectively. Ischemia induced cerebral edema was reduced in a dose dependent manner. Curcumin at 300 mg/kg, i.p. produced 50.96 +/- 6.04% reduction in edema (p < 0.05) volume. Increase in lipid peroxidation after MCAO in ipsilateral and contralateral hemisphere of brain was observed, which was reduced by curcumin (300 mg/kg, i.p.)-treatment. Decrease in superoxide dismutase and glutathione peroxidase activity was observed in ipsilateral hemisphere of MCAO animal. Curcumin-treatment (300 mg/kg, i.p.) prevented IR injury mediated fall in glutathione peroxide activity. Peroxynitrite measured using rhodamine123 fluorescence and anti-nitrotyrosine immunofluorescence indicated increased peroxynitrite formation after IR insult. Curcumin-treatment reduced peroxynitrite formation and hence the extent of tyrosine nitration in the cytosolic proteins. These results suggest the neuroprotective potential of curcumin in cerebral ischemia and is mediated through its antioxidant activity.     

线栓法制备大鼠局灶性脑缺血模型手术操作的研究进展

线栓法大鼠大脑中动脉阻塞模型被普遍认为是标准的大鼠局灶性脑缺血动物模型。该模型在制备过程中,易受到多重因素的影响,其中手术操作对模型的成功制作起着关键作用。文章从大鼠颈部主要动脉的解剖位置、手术切口位置的选择、手术左右侧别的选择,以及插线位置的选择进行了综述。     

常压氧与高压氧对成年大鼠脑缺血再灌注损伤后微血管新生影响的比较

目的:研究常压氧与高压氧对成年大鼠脑缺血再灌注损伤后微血管新生影响的差异。方法:将成年SD雄性大鼠随机分为三组:假手术组(SS组)、常压氧治疗组(NBO组)、高压氧治疗组(HBO组),每组又随机分为3、7、10天三个亚组。采用线栓法对NBO组和HBO组大鼠进行大脑中动脉栓塞(MCAO),缺血1.5小时后拔出栓子再灌注,NBO组进行常压氧治疗,HBO组进行高压氧治疗。大鼠分别在3、7、10天麻醉处死,取脑组织切片,血管内皮生长因子(VEGF)、VEGF受体-1(FLT-1)和CD34免疫组化染色,光镜观察取图和统计分析。结果:NBO各组与SS各组相比,VEGF、FLT-1和CD34阳性细胞数目均明显增多(P<0.05);HBO各组与NBO各组比较,7天、10天组VEGF、FLT-1和CD34阳性细胞数目均显著增多(P<0.05)。结论:HBO治疗较NBO治疗对成年大鼠微血管的新生更有促进作用。     

Amino Acids in Rat Striatal Dialysates: Methodological Aspects and Changes After Electroconvulsive Shock

Extracellular levels of amino acids were estimated in dialysates of the rat striatum that were collected 1, 2, and/or more than 5 days after surgery, before (resting release) and during exposure to high K concentrations (50 mM) or electroconvulsive shocks. The resting release of several amino acids (Glu, Asn, Thr, Tau, Tyr, Gly, and Ala) was higher 9 days as compared to 1 day after surgery. In the 1-day preparation the resting release correlated highly with that observed with push-pull cannulas. The correlation with the tissue content of the amino acids was high only when they were divided into two groups (putative transmitters and metabolic intermediates). High K exposure produced increased output of Ala, ethanolamine (Eam), Asp, Glu, Tau, and Gly and a decrease in the egress of Gln 1 or 2 days after surgery. The effects on Asp and Glu had disappeared, and that on Gln reversed after 4-9 days. Electrically induced convulsions produced increased output of Ala, Gln, and Eam 1 or 2 days and 2 weeks after implantation of the probe. Changes were seen not only during but also (and some cases even more prominent) after the seizure. This study shows the usefulness of dialysis to monitor extracellular transmitter amino acids in the striatum of conscious rats (also bilateral dialysis was possible) for only a limited time after implantation of the probe. The dialysis method is suitable for longer time, when metabolic changes in amino acids are to be followed. In addition to transmitter release, glycolysis can be monitored by the measurement of Ala in the dialysate.     

Glutamate Release in Experimental Ischaemia of the Retina: An Approach Using Microdialysis

A rabbit eye model of neural ischaemia is described that uses an increased pressure in the anterior eye chamber to block the capillary supply to the retina. A microdialysis probe placed very close to the retinal surface was used to monitor release of amino acids during ischaemia. A large (two- to threefold) increase in the release of glutamate and O-phosphoserine (twofold), but not of six other amino acids monitored, occurred during initial ischaemia. During reperfusion after release of intraocular pressure, much larger (five- to 10-fold) increases in the release of these amino acids were observed. Parallel ischaemic retinal tissue damage was observed. This damage was prevented by ketamine applied locally via a superfusion needle, suggesting that glutamate released during ischaemia, and particularly during reperfusion, was responsible for cell death.     

Effects of Ionotropic Excitatory Amino Acid Receptor Antagonists on Glutamate Transport and Transport-Mediated Changes in Extracellular Excitatory Amino Acids in the Rat Striatum

Abstract: This study examined the effects of intrastriatal administration of ionotropic excitatory amino acid receptor antagonists on biochemical markers of excitatory amino acid transmission in the rat striatum. High-affinity glutamate uptake was measured ex vivo on striatal homogenates 15 min after the local administration of either 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA receptor antagonist, or dl -2-amino-5-phosphonopentanoic acid (AP5), a competitive NMDA antagonist, at various doses (10–500 pmol injected). DNQX induced a dose-dependent increase in glutamate uptake rate, related to an increase in the V _max of the transport process, whereas no significant change in glutamate uptake was detected after AP5 administration. Similar results were obtained from animals subjected to excitotoxic lesion of striatal neurons by kainate administration 15 days before the injection of DNQX or AP5. In a parallel series of experiments using in vivo microdialysis we showed that DNQX (10⁻⁵ M ) in the dialysis probe diminished by ∼30–40% the increases in the concentrations of glutamate and aspartate elicited by l - trans -pyrrolidine-2,4-dicarboxylic acid (1 m M ). These data suggest that presynaptic glutamate transmission in the rat striatum may undergo facilitatory autoregulatory processes involving ionotropic non-NMDA receptors and highlight the view that transporters for glutamate may be potent regulatory sites for glutamatergic transmission.     

Neuroprotective effects of lactate in brain ischemia: Dependence on anesthetic drugs

Lactate is a major energy source for the brain, especially when glucose is not available in sufficient amounts. In the present study, we administered sodium l-lactate (250 mg/kg) to mice before or after middle cerebral artery occlusion (MCAO) to test whether lactate can be neuroprotective in brain ischemia. Permanent ischemia for 24 h caused a large hemispheric lesion and a severe loss of body weight. Administration of lactate shortly (15–30 min) before MCAO strongly reduced cell death and weight loss, but only when isoflurane was used for anesthesia. Under pentobarbital anesthesia, lactate was inactive. After transient ischemia, when isoflurane or ketamine–xylazine were used as anesthetic drugs, lactate was effective when given immediately after reperfusion. In separate experiments, we found that plasma lactate levels are also strongly influenced by anesthetic drugs. Thus, isoflurane anesthesia as well as lactate administration caused strongly increased plasma levels of lactate whereas pentobarbital anesthesia significantly reduced plasma lactate. We conclude that exogenous lactate is neuroprotective in an in vivo-model of brain ischemia, but that its action is strongly influenced by the type of anesthetic agent used.     

A novel calpastatin-based inhibitor improves postischemic neurological recovery

Calpastatin, a naturally occurring protein, is the only inhibitor that is specific for calpain. A novel blood-brain barrier (BBB)-permeant calpastatin-based calpain inhibitor, named B27-HYD, was developed and used to assess calpain’s contribution to neurological dysfunction after stroke in rats. Postischemic administration of B27-HYD reduced infarct volume and neurological deficits by 35% and 44%, respectively, compared to untreated animals. We also show that the pharmacologic intervention has engaged the intended biologic target. Our data further demonstrates the potential utility of SBDP145, a signature biomarker of acute brain injury, in evaluating possible mechanisms of calpain in the pathogenesis of stroke and as an adjunct in guiding therapeutic decision making.     

Effects of cathepsins B and L inhibition on postischemic protein alterations in the brain

The effects of selective inhibition of cathepsins B and L on postischemic protein alterations in the brain were investigated in a rat model of middle cerebral artery occlusion (MCAO). Cathepsin B activity increased predominantly in the subcortical region of the ischemic hemisphere where the levels of collapsing mediator response protein 2, heat shock cognate 70 kDa protein, 60 kDa heat shock protein, protein disulfide isomerase A3 and albumin, were found to be significantly elevated. Postischemic treatment with Cbz-Phe-Ser(OBzl)-CHN₂, cysteine protease inhibitor 1 (CP-1), reduced infarct volume, neurological deficits and cathepsin B activity as well as the amount of heat shock proteins and albumin found in the brain. Our data strongly suggests that the decrease in heat shock protein levels and the significant reduction of serum albumin leakage into the brain following acute treatment with CP-1 is indicative of less secondary ischemic damage, which ultimately, is related to less cerebral tissue loss and improved neurological recovery of the animals.     

Effect of transient focal ischemia of mouse brain on energy state and NAD levels: no evidence that NAD depletion plays a major role in secondary disturbances of energy metabolism

It has been proposed that NAD depletion resulting from excessive activation of poly(ADP-ribose) polymerase is responsible for secondary energy failure after transient cerebral ischemia. However, this hypothesis has never been verified by measurement of ATP and NAD levels in the same tissue sample. In this study, we therefore investigated the effect of transient focal cerebral ischemia on the temporal profiles of changes in the levels of energy metabolites and NAD. Ischemia was induced in mice by occluding the left middle cerebral artery using the intraluminal filament technique. Animals were subjected to 1-h ischemia, followed by 0, 1, 3, 6, or 24 h of reperfusion. During ischemia, ATP levels, total adenylate pool, and adenylate energy charge dropped to approximately 20, 50, and 40% of control, respectively, whereas NAD levels remained close to control. Energy state recovered transiently, peaking at 3 h of recovery (ATP levels and total adenylate pool recovered to 78 and 81% of control). In animals subjected to reperfusion of varying duration, the extent of ATP depletion was clearly more pronounced than that of NAD. The results imply that depletion of NAD pools did not play a major role in secondary disturbances of energy-producing metabolism after transient focal cerebral ischemia. Changes in ATP levels were closely related to changes in total adenylate pool (p<0.001). The high energy charge after 6 h of reperfusion (0.90 versus a control value of 0.93) and the close relationship between the decline of ATP and total adenylate pool suggest that degradation or a washout of adenylates (owing to leaky membranes) rather than a mismatch between energy production and consumption is the main causative factor contributing to the secondary energy failure observed after prolonged recovery.